Description:

Objective:

The objective of this research project was to test the hypothesis that exposure of animals to dibutyl phthalate (DBP) during differentiation of the reproductive system, even at relatively low concentrations, alters reproductive function as adults. This hypothesis was tested in two animal models, an amphibian, Xenopus laevis, and a non-rodent mammal, the rabbit. The former model facilitates transdermal exposure and evaluation of an easy-to-monitor, unique thyroid hormone-dependent event, the metamorphosis, while the latter facilitates longitudinal evaluations of hormones, semen parameters, and sexual capacity. Furthermore, rabbits, unlike rodents, have a relatively long infantile period of reproductive development more closely approximating the situation in humans. A dermal route of exposure is particularly pertinent to children’s vulnerability to toxic substances in the environment, since children have a greater ratio of surface area to body weight than adults.

Experiment 2. Xenopus tadpoles were exposed to 0, 0.1, 0.5, 1.0, 5.0, or 10.0 ppm DBP beginning at sexual differentiation (Nieuwkoop and Faber stage 52; 3 weeks of age) and continuing until 100% of controls metamorphosed (stage 66; 8 weeks of age). Upon necropsy at 33 weeks, 4–6% of DBP-treated frogs had only one testis and 2–4% had retained oviducts. In all DBP treatment groups compared with controls, seminiferous tubule diameter and the average number of germ cell nests per tubule were lower, and the number of tubules with no germ cells was significantly higher (p < 0.05). The percent of secondary spermatogonial cell nests significantly decreased (p < 0.05) in the 1.0, 5.0, and 10.0 ppm groups. Several lesions occurred in DBP-exposed testes, including denudation of germ cells, vacuolization of Sertoli cell cytoplasm, thickening of lamina propria of seminiferous tubules, and focal lymphocytic infiltration. Entire sections of testes containing almost exclusively mature spermatozoa were found in 1.0, 5.0, and 10.0 ppm DBP-exposed testes, indicating impairment of spermiation. Testicular hypoplasia and seminiferous tubular dysgenesis were also evident in DBP-treated frogs. Thus, sub-chronic exposure to low concentrations of DBP impairs spermatogenesis in X. laevis frogs (Lee and Veeramachaneni, 2005).

Experiment 3. Xenopus tadpoles were exposed to 0, 0.1, 0.5, 1.0, 5.0, or 10.0 ppm DBP beginning at 96 hours of age (after completion of organogenesis; Nieuwkoop and Faber stage 46) and continuing until 9 weeks of age (when 98% of solvent control animals attained stage 66; completion of metamorphosis). One week after the beginning of treatment, the percentage of mortalities in control (culture medium), solvent control, and 0.1, 0.5, 1.0, 5.0, and 10.0 ppm DBP treatment groups were 0, 1, 2, 2, 0, 32, and 87, respectively. The percentage of tadpoles that attained metamorphosis by day 60 in the corresponding groups was 81, 77, 67, 61, 14, and 0, respectively. Compared to solvent controls, DBP caused a significant delay in the rate of metamorphosis as evidenced by a low metamorphic index (hind limb length:tail length), which was reflected in a significant retardation in the average stage of development. Thus, DBP has a pronounced effect on the survival and normal progression of metamorphosis in X. laevis even when exposed after completion of organogenesis.

Residue Analysis. State-of-the-art analytical methods for the assessment of human exposure to DBP, via urinary analysis of its metabolite monobutyl phthalate (MBP), use high-performance liquid chromatography–tandem mass spectrometry (HPLC–MS/MS). This instrumentation is only available to a small number of laboratories due to its high cost. Using gas chromatography–isotope dilution mass spectrometry (GC–IDMS), which is ordinarily found in most analytical laboratories, we applied available GC–MS methods to determine DBP and MBP in biological matrices obtained from DBP-exposure studies, namely, frog embryos, culture media, and rabbit blood plasma/serum. The GC–IDMS method developed was sensitive. The limits of detection and quantification in method development, using urine as a biological matrix, were 0.2 and 0.7 ng/mL, respectively; in blood plasma/serum, limits were 0.5 and 1.6 ng/mL MBP.

MBP was detected in Xenopus embryos and culture media as early as 8 hours post-treatment indicating that DBP is metabolized to MBP by frog embryos and that MBP could be responsible for the toxicity observed in FETAX.

Analysis of blood plasma/serum of DBP-exposed rabbits revealed that levels of MBP in the progeny were highly correlated with those of their dams. By postnatal week 12, MBP was undetectable in blood plasma of rabbit progeny prenatally exposed to DBP indicating complete and rapid metabolism of DBP.

Animal Experiments: Rabbit Model

Because rabbits have a relatively long phase of reproductive development, simulating that of humans better than rodents, and because use of rabbits facilitates multiple evaluations of mating ability and seminal quality, we used this animal model. Rabbits were treated with DBP at three different periods of sexual development and maturation at a dose level known to adversely affect testicular function in rodents without causing systemic toxicity and sequelae. Dutch-Belted rabbits were exposed to 0 or 400 mg DBP/kg/day in utero (gestation days (GD) 15–29), and during adolescence (postnatal weeks 4–12) and male offspring were examined at 6, 12, and 25 weeks of age. Another group was exposed after puberty (for 12 weeks) and examined at the conclusion of exposure. The most pronounced reproductive effects were in male rabbits exposed in utero. One of the 17 male offspring in this group manifested hypospadias, hypoplastic prostate, and cryptorchid testes with carcinoma in situ-like cells. As adults, they exhibited a reduction in the number of ejaculated sperm (down 43%; p < 0.01), weights of testes (at 12 weeks; down 23%; p < 0.05) and accessory sex glands (at 12 and 25 weeks; down 36%, p < 0.01; and 27%, p < 0.05), and serum testosterone levels (at 6 weeks; down 32%, p < 0.05); a slight increase in histological alterations of the testis (p < 0.05); and a doubling in the percentage (from 16 to 30%, p < 0.01) of abnormal sperm. In the DBP group exposed during adolescence, basal serum testosterone levels were reduced at 6 weeks (p < 0.01), while at 12 weeks testosterone production in vivo failed to respond normally to a GnRH challenge (p < 0.01). In addition, the weight of the accessory sex gland was reduced at 12 weeks but not at 25 weeks after a recovery period; there was a slight increase in the percentage of abnormal sperm in the ejaculate; and 1 of the 11 males was unilaterally cryptorchid. In both of these DBP-treated groups, daily sperm production, epididymal sperm counts, mating ability, and weights of body and non-reproductive organs were unaffected. Thus, DBP induces lesions in the reproductive system of the rabbit with the intrauterine period being the most sensitive stage of life (Higuchi, et al., 2003).

Conclusions:

In summary, DBP at environmentally relevant concentrations has the potential to cause irreversible damage to amphibian populations by affecting survival, development, growth, and spermatogenesis. The magnitude of vulnerability of tadpoles to relatively low levels of DBP points to another potential contributing factor in amphibian declines. In rabbits, DBP induces lesions in the reproductive system with the intrauterine period of development being the most sensitive stage of life. The anomalies include impaired differentiation of male reproductive organs, testicular maldescent, abnormal differentiation of germ cells with lesions resembling carcinoma in situ, and aberrant spermiogenesis characterized by acrosomal dysgenesis.

As envisaged, this work resulted in the development of useful laboratory animal models to address contemporary environmental issues and their potential impact on human reproductive health as related to phthalate exposures. Furthermore, an association between developmental DBP exposures and insidious effects was established; these effects include embryonic mortality and delayed metamorphosis in frogs at relatively low-level exposures of DBP, and manifestation of testicular dysgenesis in rabbits characterized by germ cell atypia resembling carcinoma in situ and persistent acrosomal dysgenesis.

The perspectives, information and conclusions conveyed in research project abstracts, progress reports, final reports, journal abstracts and journal publications convey the viewpoints of the principal investigator and may not represent the views and policies of ORD and EPA. Conclusions drawn by the principal investigators have not been reviewed by the Agency.